Exam 2: Structural Proteins - Globular

n

-"Hill Cooperativity Coefficient" -the key to the dramatic difference in oxygen transport efficiency is Hbs cooperativity -as n changes, so does the sigmoidal shape of the O binding curve -very important as it tells you the number of bindings sites -for any protein in the world to have any cooperativity n must be greater than 1

carbon monoxide

CO can bind to the heme Fe(II) in hemoglobin to outcompete oxygen - the affinity of Hb for CO is 250 times greater than its affinity for oxygen -

advantage of sickle cell anemia

-Advantage against malaria- the disease cannot bind and be potent. -Virus cannot bind to the sickled cell, needs round cell -Interestingly, in regions with a lot of malaria there are more cases of sickle cell anemia. High as 40% in certain parts of Africa

gamma hemoglobin

-At embryonic stage initially have gamma form (flattens out after first three months) -As soon as child is born and can breathe on their own, the gamma form is no longer required so the gamma form no longer exists within first 6 months -Gamma binds to oxygen more tightly because the child does not have as much available. Due to a different amino acid sequence -Function of gamma form is to increase oxygen binding so that fetus can have enough oxygen to survive gamma hemoglobin

why does BPG bind to the cavity

-BPG fits so nicely because of ionic interactions between the negative phosphate groups and the positive Ns on K and H -All residues around it are basic, and BPG is highly negative, so ionic interactions -H bonding of course, but primary interactions are ionic

How to identify sickle cell anemia

-DNA testing -restriction enzyme digestion with MstII yields 3 fragments for a normal B gene but only 2 for the sickle cell gene

Fe (II) vs Fe (III)

-Fe(III) has a lower affinity for oxygen -Fe (II) converts to Fe(III) when it is in the presence of oxygen (it becomes oxidized) -the protein portion of Hb prevents oxidation from happening and allows oxygen to reversibly bind to the heme group -under certain conditions, when Fe(II) becomes oxidized, we call this methmyoglobin or methmoglobin

Globular proteins

-Hemoglobin: system wide carrier of oxygen that is transported through the blood by RBCs -Myosin: small monomeric molecule that acts as a secondary carrier of oxygen in the muscle tissue -IgG: immuno gamma globulin, high MW protein in immune system -actin and myosin: maintain cell cytoskeleton

If our body's production of 2,3 BPG has increased, what health concerns could resutl?

-Maybe this is a metabolic reason for a mutation in the enzyme that forms BPG so we have an excess of BPG. -If there is no oxygen binding to Hb at all then there will be no oxygen left for tissues -Initially it is good because tissues will be getting lots of oxygen, until there is none left -Usually the R→ T state is equal and R is quite stable -If this perpetually moves in one direction then R state becomes impossible so oxygen binding would be low and then none would be able to be released so it would be fatal

When Mb and Hb are present in the same solution, which would compete more effectively for oxygen? What if Mb and Hb solutions were separated by a semipermeable membrane?

-Myoglobin would compete more effectively there is less resistance because it does not have cooperativity when oxygen is binding to it -look at graphs and know why -Doesn't matter, same water solution so it's still myoglobin -Myoglobin always has a higher affinity for oxygen

Hb Oxygen Transport and Cooperativity

-One ligand of molecular oxygen binds to each of the four iron atoms sequentially, and the affinity of each of the four sites changes as they become occupied w O2 -Cooperativity: When one subunit of hemoglobin changes because of oxygen binding, a change is induced in the next, and the next, and etc. ultimately a change in one subunit changes the entire structure -Hb exhibits low affinity for the first oxygen molecule to bind. the first forward reaction is very slow, not at all favored. The oxygen meets resistance as the Hb does not want it to bind -the subsequent oxygen bindings increase in speed and ease, as more O binds the Hb gets used to it and by the time it gets to the fourth O2 the protein does not want to lose oxygen. -the protein undergoes a complete conformation change from the T state (deoxygenated) to the R state (oxygenated) -know graph

Why is it beneficial for Hb to exhibit such different affinities for oxygen?

-We need controlled binding and release so that we don't just have all or nothing oxygen, but can meet according to needs and so that everywhere can get oxygen -When more oxygen is required or less oxygen is required chemical reactions occur in body so more oxygen can be released when needed, ie during exercise or at high altitudes

how to treat CO Poisoning

-We want to get rid of CO and replace with O2 -Put patient in oxygen tent -O2 tube and atmosphere high in oxygen -This will force CO out (even though it has a higher affinity) -Forced to kick out CO

which condition is worse in terms of yielding the lowest levels of functional hemoglobin: inactivation of half the globin subunits due to interaction with CO or due to low iron levels?

-Worse for somebody that is anemic because they do not have the CO defense mechanism so they cannot push teh Hb to bind to more oxygen, it is just always slow -be able to explain this graphically. Look at sloped -however, under fatal CO poisoning conditions, the binding interaction with CO can become irreversible

apoprotein vs holoprotein

-apoprotein is the protein without the prosthetic group, so just the protein part -holoprotein is the entire protein including the prosthetic group and iron

BPG binding

-binds inside the cavity between the four subunits of Hb -only binds to deoxygenated (T state) Hb, when there is room in the cavity -When 1+ O2 molecules are bound, it cannot fit into the smaller cavity. -therefore, binding of O2 and BPG is mutually exclusive (one or the other). Both cannot bind at the same time

role of distal histidine

1). The position of E7 prevents O2 from binding too tightly to the iron atom. Maximum binding strenght is achieved when the three atoms (Fe-O=O) form a linear sequence (which they want to do). However, the distal histidine prevents this from occurring, and the diatomic oxygen binds in a bent configuration. This ensures that oxygen is not bound too tightly and can be released to the tissues 2) Carbon monoxide binds to the iron atom with a higher affinity than oxygen because it is more polar. Even in low [], it will displace the oxygen and bind tighter to Fe. Distal His prevents this. It acts almost like a gate, slowing down the binding of CO to heme and reducing its toxicity to our bodies. If we did not have E7 even a tiny amount of CO would be toxic. Also, CO also binds in a bent configuration, this weakens the attraction so that over time the CO will dissociate and one can recover

role of proximal histidine

-in the unbound state, the iron atom is slightly proximal (above) the plane of the protoporphyrin -as oxygen binds to the distal side of the ring, it pulls the iron atom about 0.2 angstroms closer to the plane of the ring -although this distance is small, the movement is amplified, causing significant shifts throughout the tertiary structure of the protein -the angle at which porphyrin binds is changed, due to the proximal histidine

BPG and oxygen binding Hb curve - Effect of 2,3-BPG on Cooperativity

-increasing the concentration of BPG shifts the plot of oxygen binding to Hb to the right, as it reduces the oxygen affinity. This means the deoxygenated form is stabilized, pushing the plot to the right. -the dissociation of O2 in the peripheral tissues is increased

iron ion

-iron ion is the actual binding site for oxygen molecules -converts between teh Fe2+ (ferrous) state and the bound Fe3+ (ferric ion) state. -when oxygen binds to the iron, the iron is pulled into the plane of the ring so the ring becomes more planar as well

structure of myoglobin

-largely a-helical in structure, with a total of 8 separate and distinct a-helical secondary structures (all alpha + turns) -153 amino acid residues in a highly folded and compact structure -the heme group is bound in a hydrophobic cleft and held there by hydrophobic and van der Waals interactions and H-bonds -prosthetic group is proto-heme. The proto-heme in the hydrophobic heme pocket is essential for the reversible oxygen binding -measurements: 45x35x25 Å with 70% alpha helix content

Blood pH and Hb oxygen affinity

-lower pH decreases oxygen affinity -this automatically releases oxygen in peripheral tissues where active respiration has produced increased levels of CO2, resulting in lower pH caused by carbonic acid -"Bohr Effect" -the result of the Bohr Effect is to deliver more total oxygen between the lungs and the peripheral tissues

polycythemia

-mutations that increase Hb's affinity for oxygen tend to lead to increased numbers of erythrocytes to compensate for the less than normal amount of oxygen released in the tissues. -this condition is known as polycythemia -can result in red skin

Hemoglobin structure

-nearly spherical with a 55 Å diameter and a MW of 64.45 kD -has 4 protein subunits, 4 protoporphyrins, and 4 iron atoms -each hemoglobin molecule can transport 4 oxygen molecules (one per Fe atom) -dimer of dimers -two alpha subunits have 141 amino acids -two beta subunits have 146 residues

heme

-prosthetic group of myoglobin -each myoglobin molecule has one -a protoporphyrin IX and a central Fe iron -collectively called "heme" -held in place by hydrophobic interactions to the nonpolar interior region of the protein -not attached by any covalent linkages, and can be removed resulting in the apoprotein. easily removed by changes in conditions -an iron ion fits perfectly into the center of the protoporphyrin, chelated by 4 N atoms of a tetrapyrrole ring system. -heme group gives it FUNCTION-without it hemoglobin and myoglobin cannot hold iron, and will just be useless protein

myosin and hemoglobin

-proteins that evolved to carry oxygen in the blood streams of vertebrate organisms -this is an important bio innovation that enabled a reduced viscosity compared to circulatory systems in which oxygen carriers were directly dissolved in blood plasma

Sickle Cell Anemia

-results from a single amino acid change: Glu --> Val at position #6 of the beta chains, which is at the surface of the protein -this is a genetic disease that drastically impedes oxygen transport and is manifested as distorted "sickled" erythrocyte shapes -aka HbS, Hemoglobin S

what happens because of the amino acid change in sickle cell anemia?

-this point mutation completely changes the structure because you are going from a hydrophilic to hydrophobic residue. -Glu probably had ionic interactions with adjacent basic groups, but now these are completely disrupted when Val, neutral and hydrophobic is there -the driving force for the formation of fibrous proteins is hydrophobic interactions. So, this is a major disruption. When it becomes hydrophobic we start to get fibers, which are long and stringy so we end up getting sickle shaped red blood cells -Self assembly completely changes and it becomes totally fibrous, and has long polymeric forms, -at low [O2], HbS molecules stick to one another forming long polymeric chains. 0these long polymeric forms are locked into deoxy forms, and while polymerized CANNOT BIND OXYGEN -There is less space so it is more locked into deoxy forms and cannot bind to oxygen -Symptom severity varies form mild to sever and need blood transfusions every three weeks

What happens when we increase in altitude? Then go back?

-when we change altitude, during the initial few days, the body responds by adjusting Hb cooperativity. This can be accomplished by changing the concentration of 2,3-BPG in the blood. -As you go up in altitude you increase BPG production to promote release of oxygen (by lowering Hb affinity for oxygen) so that the tissues can get more oxygen If you were to breathe normally your tissues would not be getting enough oxygen When you go back to lower altitudes, initially your BPG production will still be high but eventually goes back down It can be detrimental if levels always remain high

BPG

2,3-Biphosphoglycerate -a small, highly polar molecule -responsible for much of the cooperativity in Hb

formation of BPG

3C compound that is a derivative of glycerol, converted during glycolysis -glycolysis becomes pyruvate -during the 5th or 6th step we get hte formation of 1,3-BPG, which is essentially the same but phosphates at dif positions. -can convert back and forth from 1,3 to 2,3 depending on the conditions needed: -2,3 BPG made during conditions when higher O2 release is essential -1,3 goes on and breaks down into pyruvate, but can go through this reversal enzymatic reaction for form 2, 3

myo

=muscle -myoglobin is the oxygen carrier in tissue, and the most prominent tissue int he body is muscle

what is a prosthetic group>

A non-protein component that forms a permanent part of a functioning protein molecule -parts of protein that are not amino acids -metal ions (coenzymes) -organic molecules that are essential for protein function

Fetal hemoglobin

Fetal hemoglobin's gamma chains have serine in place of histidine 143 (adult beta chains) -this serine is near the binding site for 2,3-BPG and reduces the affinity of fetal Hb for 2,3-BPG -this results in an increased affinity for oxygen -therefore, a fetus can effectively draw oxygen across the placental membrane -O2 flows from maternal oxyhemoglobin to fetal deoxyhemoglobin

graph of hemoglobin binding to oxygen vs Mb

Hb has sigmoidal shape!!! means that there is cooperativity involved Mb has a hyperbolic shape!! means that there is no cooperativity involved. Reflects hat Mb has 10x higher affinity for O2 than Hb and only one binding site so binds easier.

What if a solution of O2 saturated Hb were placed on one side of the membrane in a solution of free (unbound) Mb were placed on the other side of the membrane? (Po2 = 10 Torr)

IF anything it will be even faster because Hb will not be competing since it is already saturated -O2 comes off Hb to go to Mb -at this PO2, Mb has a MUCH higher affinity for O2

Hb and CO2

In addition to its role in oxygen transport, Hb also transports CO2 from the tissues back to the lungs for disposal -CO2 binds to the terminal amino groups by forming carbamates -in the alveolae, the equilibrium shifts and the carbamates revert to free amines, releasing CO2

allostery/allosteric stie

When you make one change at one position, the whole thing is affected -Usually when you have an allosteric site the plot will be sigmoidal -Not directly related to the actual site, but sites that have domino affect -If the plot is sigmoidal, there is definitely allosteric sites -Hemoglobin has a allosteric site

why does histidine play such an important role?

aromatic ring systems tend to stack, so these histidines help iron stay attached -very very important in helping everything stay together -Histidine has affinity for metals, so anywhere you have histidines (especially how they are positioned in these two positions) it plays a very important role

Mb and Hb binding to oxygen at different concentrations

at lower concentrations of oxygen (as int eh capillary Mb has higher affinity for O2 than Hb -but in somewhere where [O2] is very high, such as in the lungs, both will be saturated.

Function of BPG

binding of BPG reduces Hb's affinity for O2 -so in conditions where the cells/tissues need more oxygen, BPG will promote its release to ensure they get oxygen.

methmyoglobin or methmoglobin

certain mutations at the O2 binding site of either the a or B chain favor the oxidation of Fe(II) to Fe (III). -individuals carring hte resulting methemoglobin subunit exhibit cyanosis, a bluish skin color, due to the presence of methemoglobin in their arterial blood. -these hemoglobins have reduced cooperativity (Hill constant is about 1.2 compared) -

Adult vs Embryonic Hemoglobin

embryonic hemoglobin exhibits a higher affinity for oxygen than adult hemoglobin -Fetus cannot breathe on its own and is completely dependent on the mother's oxygen. Whatever is coming in through the placental membrane is what the fetus gets -different abundances of different forms reflects the fetus' increased need for hemoglobin with high oxygen affinity

Hill equation

explains the affinity of Hb towards oxygen

Hb and oxygen solubility

hemoglobin is located in erythrocytes, where it greatly increases oxygen solubility, facilitating as much as 68 times higher oxygen concentrations than in water alone -each erythrocyte contains about 300 million hemoglobin molecules

histidine-iron coordination in prosthetic group

iron ions are hexadentate, so each has six coordination sites. -one of these forms a coordinate covalent bond to a N in histidine F8 (proximal) -another histidine (E7, distal) is close to the 6th coordination position

beta hemoglobin

not very important until birth, but then it maximizes and stays (hemoglobin is beta form)

iron deficient anemia Hb

only 50% of heme cofactors occupied w Fe2+

Zeta and epsilon hemoglobin

only required in first few months as a fetus

alpha hemoglobin

present throughout fetal growth and after birth, indicating it plays an important role in oxygen bonding (why normal Hb is aB)

red color of blood

results from the oxygenated forms of myoglobin and hemoglobin, which is caused by a change in the electrical state of the bound heme prosthetic group

Hb dialysis expt and conclusions

simple dialysis revealed a key mechanism in Hb cooperativity: -when erythrocyte contents are dialyzed, the Hill coefficient drops to n=1! When the permeate is remixed with hemoglobin, the value of n returns to normal (~3.3) conclusions: 1). Hb has the ability to go back to normal when conditions are returned to normal 2). RBCs have other chemicals that can affect Hbs affinity for oxygen (ie H+, bicarbonate, BPG). -The binding is increasing, so the curve goes left and you have to be removing things that delay the binding of hemoglobin -think about the Bohr effect, which reduces binding of oxygen so it can be released from tissues. what else is involved that is increasing binding when dialysed?

hemoglobin vs myoglobin

size -myoglobin is much smaller. Single subunit/polypeptide, and only has tertiary structure. -hemoglobin is much bigger because it is carrying oxygen throughout the entire system. Vertebrate hemoglobins are tetrameric -both have a similar folding pattern, so they are thought to have evolved from the same ancestor -both have roles in binding to and transporting oxygen structural: -both the a and B subunits of Hb have structural characteristics similar to Mb. -if you compare the amino acid sequences of Hb a and B, and myoglobin, only 27% of the residues are identical. It is likely that this 27% is what is involved in the oxygen carrying ability f'n as they were conserved as Hb and Mb evolved/diverged

p50

the amount of oxygen that binds to 50% of oxygen binding sites -very important info in pathological studies

what causes cooperativity in Hb?

the key to understanding this is understanding the changes in Hb's tetrameric structure when O2 binds

R and T states of Hb

the transition fromt eh T state to the R state shifts the subunit pairs, affecting certain ion pairs. Most noticeably, the His HC3 residues at the carboxyl termini of hte B subunits, which are involved in ion pairs in the T state, rotate in the R state toward the center of the molecule, where they are no longer in ion pairs. Another dramatic result of the T--> R transition is a narrowing of the pocket between the B subunits -when O2 is not bound, the T state structure allows 4 stabilizing ion pairs to form (one in each of the 4 subunits) -binding of oxygen to the iron pulls the helix containing Aspartate 94, disruption the ion pair. Therefore, energy must be added to the T state to allow it to bind O2. In other words, the T state has a lower affinity for O2 compared to the R state, which lacks these ion pairs

Why do the binding curves for the CO exposed Hb and the iron deficient anemia Hb max out at Y=0.5 (when normal Hb goes almost to 1.0)?

when Hb starts binding to CO, it changes conformation of Hb such that it pushes it to bind to more oxygen. This is the bodies defense mechanism (only works under mild CO exposure) -at 50% it gets saturated and nothing else can bind -with anemia, you do not haev enough iron so you will never get to that level

methemoglobin

when iron is in the Fe3+ form, it cannot bind to oxygen, and if it stays here for a long time it becomes problematic as body tissues cannot get oxygen. -Fe is in oxidized form -ex: brown meat at grocery store has been sitting for a while and is very oxidized

What are the changes that occur when O2 binds?

when the first O2 molecule binds to one of the 4 heme groups a number of structural changes occurs: -the movement of hte Fe atom into the heme plane also draws in teh F8 (proximal) histidine, leveraging a big change in its subunit -the alpha and beta groups rotate ~15 degrees with respect to one another, disrupting non-covalent linkages between its neighboring subunits and breaks salt bridges -the open channel int eh center of the subunits becomes much smaller, bringing the beta chains much closer than before. *these structural changes INCREASE affinity for oxygen in the remaining 3 subunits*